A strain Bacillus subtilis subsp.subtilis CEGF012 and a culture method thereof

Abstract

The present application relates to a strain Bacillus subtilis subsp.subtilis CEGF012 and a culture method thereof, the strain Bacillus subtilis subsp.subtilis CEGF012, the preservation number of which is CCTCC NO: M 20241702, and the preservation date of which is July 31, 2024; and the culture method comprises the step of inoculating a seed liquid of CEGF012 into a solid culture medium for culture. Bacillus subtilis subsp.subtilis The activity of the strain is as high as 9.16 x 10 9 CFU / g, and the strain can grow well in a culture medium with plant-based refractory protein with a protein content of 15% to 30% as a nitrogen source, thereby being beneficial to reducing the cost of culture with protein peptone as a nitrogen source.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial fermentation, and more particularly to a strain Bacillus subtilis subsp. subtilis CEGF012 and a culture method thereof. BACKGROUND

[0002] Bacillus subtilis is a kind of Bacillus, which is widely used in food, feed additive, seed protection, biological control, biological preservation, animal breeding, crop disease and pest control and biological fertilizer production fields. At present, the production methods of Bacillus subtilis include solid state fermentation method and liquid state fermentation method.

[0003] However, the commonly used typical culture medium usually takes starch, glucose and the like as carbon source, and takes beef extract, peptone and the like as nitrogen source, which has high cost and is difficult to popularize and apply in industry. In order to reduce the cost, industrial culture medium is generally used for large-scale production in industry, which is easy to cause the strains screened by the typical culture medium to be difficult to achieve the expected effect on the industrial culture medium. Therefore, screening Bacillus subtilis with high activity and capable of growing on the industrial culture medium is conducive to promoting its application in industry.

[0004] In addition, Bacillus subtilis strains have diversity, and the suitable culture medium and fermentation conditions thereof are different for different Bacillus subtilis strains. For example, the conventional fermentation technology cannot achieve the expected effect of high-density fermentation for the Bacillus subtilis YXSY-01 in patent CN117568235B. Therefore, it is necessary to study the suitable culture method for different Bacillus subtilis in order to promote its large-scale industrial production. SUMMARY

[0005] In view of the above defects or improvement needs of the prior art, the present application provides a strain Bacillus subtilis subsp. subtilis CEGF012 and a culture method thereof, which aims to separate and purify microorganisms from the root part of Populus euphratica forest in the Taklimakan Desert in Xinjiang, and screen out a high-activity strain, which is identified as a new strain of Bacillus subtilis, named Bacillus subtilis subsp. subtilis CEGF012, with a preservation number of CCTCC NO: M 20241702. The strain can be cultured by using low-cost plant-based refractory protein as nitrogen source, thereby solving the technical problem that the strains screened by the typical culture medium are difficult to achieve the expected effect in the industrial culture medium.

[0006] To achieve the above object, according to one aspect of the present application, there is provided a high-activity Bacillus subtilis subsp. Subtilis CEGF012, having a preservation number of CCTCC NO: M 20241702 and a preservation date of July 31, 2024, which can be cultured using an industrial culture medium and has high activity.

[0007] According to another aspect of the present application, there is also provided a microbial agent comprising the Bacillus subtilis subsp. Subtilis CEGF012.

[0008] According to another aspect of the present application, there is also provided a culture method of Bacillus subtilis subsp. Subtilis CEGF012, comprising the step of inoculating the seed liquid of Bacillus subtilis subsp. Subtilis CEGF012 having a preservation number of CCTCC NO: M 20241702 into a solid culture medium for culture.

[0009] Preferably, in the culture method, the nitrogen source in the solid culture medium comprises plant protein having a protein content of 15% to 30%, and the mass ratio of the carbon source to the nitrogen source is 50 to 83: 17 to 50.

[0010] Preferably, in the culture method, the mass ratio of the carbon source to the nitrogen source in the solid culture medium is 50 to 75: 25 to 50.

[0011] Preferably, in the culture method, the seed liquid of Bacillus subtilis subsp. Subtilis CEGF012 is prepared by the following method:

[0012] The Bacillus subtilis subsp. Subtilis CEGF012 is inoculated into LB liquid culture medium at an inoculation amount of 2%, and after 3 generations of recovery and propagation, the seed liquid of Bacillus subtilis subsp. Subtilis CEGF012 is obtained.

[0013] Preferably, in the culture method, the inoculation amount, the culture temperature and the culture time are determined according to the following prediction model based on the preset biomass of Bacillus subtilis subsp. Subtilis CEGF012:

[0014] ;

[0015] In the formula, Y is a preset biomass, counted by colony number 10 7 CFU / g; X1 is an inoculation amount, counted by %; X2 is a culture temperature, in ℃; and X3 is a culture time, in h.

[0016] Preferably, the culture method has the following culture conditions: the inoculation amount of the seed liquid of Bacillus subtilis subsp.subtilis CEGF012 is 4% to 10%, the culture temperature is 35 to 40 ℃, and the culture time is 24 to 36 h.

[0017] Preferably, the culture method has the following culture conditions: the inoculation amount of the seed liquid of Bacillus subtilis subsp.subtilis CEGF012 is 6% to 7%, the culture temperature is 36 to 37 ℃, and the culture time is 28 to 29 h.

[0018] Preferably, the culture method has the following culture conditions: the mass ratio of the carbon source to the nitrogen source in the culture medium is 1:1.

[0019] Overall, compared with the prior art, the above technical solutions conceived by the present application can achieve the following beneficial effects:

[0020] The strain Bacillus subtilis subsp.subtilis CEGF012 provided by the present application is isolated and purified from the root of a poplar forest in the Taklimakan Desert in Xinjiang, and is screened by using a specific screening medium prepared from an organic matter with a cellulose content of more than 10% and a protein content of less than 10%. The strain with high activity is screened out, and the activity is as high as 9.16×10 9 CFU / g. The strain is identified as a new strain of Bacillus subtilis, and is named Bacillus subtilis subsp.subtilis CEGF012. Compared with other Bacillus subtilis, the strain can grow well on a culture medium with plant-degradable protein as a nitrogen source.

[0021] In addition, the culture method provided by the present application can simulate the case of large-scale culture by using an industrial culture medium in actual application, and plant protein with a protein content of more than 15% is added to the culture medium as a nitrogen source. Not only can the cost of large-scale culture be reduced, but also it is found that plant-degradable protein with a protein content of 15% to 30% as a nitrogen source can significantly improve the activity of Bacillus subtilis subsp.subtilis CEGF012, so that high-efficiency and low-cost culture of Bacillus subtilis subsp.subtilis CEGF012 can be realized, which is conducive to promoting large-scale production. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Effect of different nitrogen sources on the activity of Bacillus subtilis subsp.subtilis CEGF012;

[0023] Figure 2 Effect of the ratio of chickpea to carbon source on the activity of Bacillus subtilis subsp.subtilis CEGF012;

[0024] Figure 3 Response surface analysis of inoculum size and culture time;

[0025] Figure 4 Response surface analysis of inoculum size and culture temperature;

[0026] Figure 5 Response surface analysis of culture temperature and culture time. DETAILED DESCRIPTION

[0027] In order to further illustrate the technical means and effects taken by the present application to achieve the predetermined purposes, the specific embodiments, structures, features and effects according to the present application are described in detail below in combination with the drawings and preferred embodiments.

[0028] The inventors isolated and purified a bacterial strain from the root part of Populus euphratica forest in the Taklimakan Desert, and screened a strain with high activity and good activity consistency from the isolated strain. The strain was identified as a new strain of Bacillus subtilis subsp.subtilis by 16S rRNA, named Bacillus subtilis subsp.subtilis CEGF012, and preserved in the China Center of Type Culture Collection, located at No.299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, on July 31, 2024 (preserved in Wuhan University Preservation Center), with the preservation number CCTCC NO: M 20241702 and the preservation date July 31, 2024.

[0029] Further, the influence of different nitrogen sources, inoculation amount, culture temperature and culture time on the activity of Bacillus subtilis subsp. subtilis CEGF012 is studied, and the results show that the strain can grow well in a culture medium with plant protein with a protein content of 15% to 30% as the nitrogen source, and can be used to replace protein peptone in a typical culture medium as the nitrogen source, such as chickpea as the nitrogen source. The growth of Bacillus subtilis subsp. subtilis CEGF012 can be significantly promoted by preparing a solid culture medium according to the mass ratio of carbon source to nitrogen source of 50 to 83:17 to 50, and preferably preparing a solid culture medium according to the mass ratio of carbon source to nitrogen source of 50 to 75:25 to 50.

[0030] Based on this, the present application provides a high-activity strain Bacillus subtilis subsp. Subtilis CEGF012, which has a preservation number of CCTCC NO: M 20241702 and a preservation date of July 31, 2024. The strain can grow well in a culture medium with high-fiber low-protein organic matter as the carbon source and plant protein with a protein content of more than 15% as the nitrogen source. The high-fiber low-protein organic matter is organic matter with a cellulose content of more than 10% and a protein content of less than 10%. In some embodiments, plant-degradable protein can be used as the nitrogen source, and the plant-degradable protein is plant protein with a fiber content of 5% to 20% and a protein content of more than 15%, such as chickpea.

[0031] In addition, the present application also provides a bacterial agent comprising Bacillus subtilis subsp. Subtilis CEGF012 as described in the present application.

[0032] In addition, the present application also provides a high-efficiency culture method for Bacillus subtilis subsp. subtilis CEGF012, which comprises the step of inoculating the seed liquid of Bacillus subtilis subsp. subtilis CEGF012 as described in the present application into a solid culture medium for culture.

[0033] The nitrogen source in the solid culture medium comprises plant protein with a protein content of more than 15%.

[0034] Preferably, the nitrogen source in the solid culture medium comprises plant-degradable protein with a protein content of 15% to 30%, and the mass ratio of carbon source to nitrogen source is 50 to 83:17 to 50; more preferably, the mass ratio of carbon source to nitrogen source is 50 to 75:25 to 50.

[0035] In some embodiments, the Bacillus subtilis subsp. subtilis CEGF012 seed solution is prepared according to the following method:

[0036] The purified Bacillus subtilis subsp. subtilis CEGF012 was inoculated into LB liquid medium at a 2% inoculation rate for activation. After three generations of culture on a shaker at 37°C, the seed culture of Bacillus subtilis subsp. subtilis CEGF012 was obtained.

[0037] Preparation of LB liquid medium (1L): Add 10g tryptone, 5g yeast extract, and 10g NaCl to 950mL ddH2O. Adjust the pH to 7.0 with 1M NaOH, bring the volume to 1L, autoclave at 121℃ for 20min, and store at 4℃.

[0038] Furthermore, the appropriate inoculum size, culture temperature, and culture time can be calculated and determined according to the following prediction model based on the preset biomass of Bacillus subtilis subsp. subtilis CEGF012:

[0039] ;

[0040] In the formula: Y is the preset biomass, based on a colony count of 10-1. 7 The data is expressed as CFU / g; X1 is the inoculum amount (%), X2 is the culture temperature (°C), and X3 is the culture time (h).

[0041] In some embodiments, the culture conditions are as follows: the inoculum size of Bacillus subtilis subsp. subtilis CEGF012 seed culture is 4%–10%, the culture temperature is 35–40°C, and the culture time is 24–36 h; preferably, the inoculum size is 6%–7%, the culture temperature is 36–37°C, and the culture time is 28–29 h; more preferably, the mass ratio of carbon source to nitrogen source in the solid culture medium is 1:1, and the nitrogen source is selected from chickpeas. In this invention, the carbon source of the culture medium may also include organic matter with a cellulose content of more than 10% and a protein content of less than 10%.

[0042] Using this culture method, with an inoculum size of 6%–7% of the Bacillus subtilis subsp. subtilis CEGF012 seed culture, a culture temperature of 36–37℃, and a culture time of 28–29 h, the colony count of Bacillus subtilis subsp. subtilis CEGF012 can reach 8 × 10⁻⁶. 9CFU / g or higher.

[0043] The following are examples.

[0044] Example 1: Isolation, purification, and screening of strains

[0045] 1. Isolation and purification of strains

[0046] Sample source: Roots of poplar forests in the Taklamakan Desert, Xinjiang

[0047] Separation material: Soil

[0048] Separation method: Take 500g of soil sample from the root base of Populus euphratica forest in the Taklamakan Desert of Xinjiang. Use the quartering method to take 10g of the sample and place it into 90mL of phosphate-buffered saline (PBS) containing glass beads. After shaking on a shaker for 10min, take 1mL of each sample and serially dilute to 10⁻⁶. -4 10 -5 10 -6 10 -7 Take 0.1 mL of 10 -4 10 -5 10 -6 10 -7 The diluted solutions were spread onto LB agar plates, with three replicates for each gradient, and incubated upside down at 30°C. Counting and observation were performed every 3 days, and incubation was stopped after 6 days. Colonies were classified and counted according to morphology, and colonies with different morphologies were selected and numbered. After purification, the colonies were stored at -80°C.

[0049] 2. Screening for highly active strains

[0050] Conventional microbial culture typically uses typical culture media (such as those containing glucose, beef extract, peptone, etc.). In this embodiment, typical culture media are first used for strain culture, isolation, and purification, and then specific screening media are used for screening. The screening media uses high-fiber, low-protein organic matter as a carbon source to simulate a culture medium that is difficult for microorganisms with high-fiber, low-protein content to utilize, and to screen strains with high activity in practical applications.

[0051] In this embodiment, the organic matter rich in dietary fiber can be selected from Cistanche deserticola, which contains 10%~30% fiber, 5%~20% polysaccharide, and 2%~8% protein. The screening of highly active strains is as follows:

[0052] (1) Preparation of screening culture medium: Soak dried Cistanche deserticola until saturated, filter out excess water, weigh 50g of wet Cistanche deserticola and crush it into soybean size, add water to the substrate moisture content to 20%, and follow (V 水 :m 湿肉苁蓉)*100% calculation, high-temperature sterilization at 121℃ for 20 min.

[0053] (2) Cultivation: Take out each strain isolated in Example 1 from the -80℃ refrigerator, inoculate them into LB liquid medium for activation, inoculate at an inoculation amount of 2%, and cultivate for 3 generations at 37℃ and 200rpm on a shaker. Then, inoculate fresh fermentation broth into the screening medium at an inoculation amount of 6% of the wet weight of the Cistanche deserticola screening medium, stir evenly, and place in a 37℃ incubator for static cultivation for 24h.

[0054] (3) Strain screening: High-activity strains were screened using the plate count method. Specifically, 0.1g of the fermentation product after 24h of culture in step (2) was placed in 9mL of PBS containing steel beads, vortexed for 1min, and 1mL was taken for 10 saturation tests. -4 10 -5 10 -6 10 -7 Serial dilution. A certain amount of the diluted solution was spread onto LB agar plates and incubated at 37°C for 1 day. The number of colonies on the plates was counted. Based on the dilution and the volume of liquid used to spread the solution, the number of viable bacteria in the original bacterial solution was calculated. The results are as follows:

[0055] Table 1 Comparison of activities of different strains

[0056]

[0057] The strain with the highest activity was identified as Bacillus subtilis subsp. subtilis, corresponding to number NA4. Identification using 16S rRNA revealed this strain to be a novel Bacillus subtilis strain, named Bacillus subtilissubsp.subtilis CEGF012. The purified and isolated strain was deposited at the China Center for Type Culture Collection (CCTCC), No. 299, Bayi Road, Wuchang District, Wuhan, Hubei Province (Wuhan University Collection Center), with accession number CCTCC NO: M20241702, and deposit date July 31, 2024.

[0058] Example 2 Effect of fermentation conditions on the activity of CEGF012 in Bacillus subtilis subsp. subtilis

[0059] Typical culture media often use beef extract and peptone as nitrogen sources, but these are costly for large-scale cultivation. In this example, a lower-cost plant protein was used as the nitrogen source to study the effects of culture medium and culture conditions on the activity of Bacillus subtilis subsp. subtilis CEGF012. The plant protein was selected from soybean meal, black beans, chickpeas, etc., wherein the soybean meal contained 15%~20% cellulose and 30%~50% protein, the black beans contained 10%~15% dietary fiber and 30%~36% protein, and the chickpeas contained 6%~10% cellulose and 19%~22% protein.

[0060] (1) Effect of nitrogen source on the activity of strain Bacillus subtilis subsp. subtilis CEGF012

[0061] Preparation of culture medium: Soaked soybean meal, black beans, chickpeas, potassium nitrate, and ammonium nitrate were used as nitrogen sources, respectively. Carbon sources were added to the culture medium at 1% of their mass. Water was added to bring the substrate moisture content to 25% (v). 水 :m 湿培养基 (×100%), autoclaved at 121℃ and 0.1MPa for 20 min to obtain the culture medium.

[0062] Inoculation and Culture: Activated Bacillus subtilis subsp. subtilis CEGF012 seed culture was inoculated at 6% of the wet weight of the culture medium and incubated statically at 37℃ for 24 h. The effect of nitrogen source on the activity of this strain was studied, and the results are as follows: Figure 1 As shown. The seed culture of Bacillus subtilis subsp. subtilis CEGF012 was prepared according to the following method:

[0063] Take out the purified Bacillus subtilis subsp. subtilis CEGF012 stored at -80℃, inoculate it into LB liquid medium at a 2% inoculum, activate it, and culture it for 3 generations at 37℃ and 200 rpm on a shaker. The fermentation broth is the seed culture.

[0064] Preparation of LB liquid medium (1L): Add 10g tryptone, 5g yeast extract, and 10g NaCl to 950mL ddH2O. Adjust the pH to 7.0 with 1M NaOH, bring the volume to 1L, autoclave at 121℃ for 20min, and store at 4℃.

[0065] Depend on Figure 1The results showed that there was no significant difference in the activity of the strain when soybean meal, black beans, or potassium nitrate were used as nitrogen sources. However, compared with soybean meal, black beans, potassium nitrate, or ammonium nitrate as nitrogen sources, chickpeas, which have a lower protein content, significantly increased the colony count of Bacillus subtilis subsp. subtilis CEGF012 and significantly promoted its growth. Therefore, chickpeas are the preferred nitrogen source for the culture of Bacillus subtilis subsp. subtilis CEGF012.

[0066] (2) Effect of nitrogen and carbon source ratio on the activity of strain Bacillus subtilis subsp. subtilis CEGF012

[0067] Using chickpeas as the nitrogen source, the carbon source and nitrogen source were mixed at mass ratios of 100:0, 99:1, 90:10, 83:17, 75:25, 50:50, and 0:100, respectively, so that the percentage of carbon source in the total mass of the two sources was 100%, 99%, 90%, 83%, 75%, 50%, and 0%. Water was added to make the substrate moisture content 25%. 水 :m 湿培养基 (×100%), autoclaved at 121℃ and 0.1MPa for 20 min to prepare the culture medium. After cooling, inoculate the seed culture of Bacillus subtilis subsp. subtilis CEGF012, which has been revived and propagated for 3 generations, at a 6% inoculation rate of the wet weight of the culture medium. Incubate at 37℃ for 24 h to study the effect of nitrogen and carbon source ratio on the activity of the strain. The results are as follows. Figure 2 As shown.

[0068] Depend on Figure 2 It can be seen that, compared with chickpea culture alone, the viable count of Bacillus subtilis subsp. subtilis CEGF012 was significantly increased when cultured in a medium prepared with a carbon source to chickpea mass ratio of 50~83:17~50. This indicates that the two can synergistically promote the growth of Bacillus subtilis subsp. subtilis CEGF012 within this ratio range, and that a medium prepared with a carbon source to chickpea mass ratio of 50~75:25~50 is preferred for culture.

[0069] Example 3: Optimization of the culture process for Bacillus subtilis subsp. subtilis CEGF012

[0070] Based on the single-factor preliminary experiment, a response surface optimization experiment was conducted. Three factors with significant influence were selected: inoculum size (factor A), culture temperature (factor B), and culture time (factor C). The biomass of strain Bacillus subtilis subsp. subtilis CEGF012 was used as the evaluation index. The Box-Behnken response surface optimization experiment with three factors and three levels was designed using DesignExpert 8.0.6 software, as shown in the table below.

[0071] Table 2 Factor Levels in the Box-Behnken Experiment

[0072]

[0073] Based on the optimization results of the single-factor experiment, a three-factor, three-level Box-Behnken response surface methodology experiment was conducted according to the Design-Expert Version 8.0.6 experimental design. The results are shown in the table below.

[0074] Table 3 Box-Behnken test protocol and results

[0075]

[0076] The table above contains 17 experimental points, with the center point (6% inoculum, fermentation at 37℃ for 36 hours) being repeated 5 times to detect experimental error. Standard polynomial regression was then used for analysis and fitting, and response surface methodology was applied to the response values ​​obtained from the 17 experimental points. The results are as follows: Figure 3 , Figure 4 and Figure 5 As shown. The analysis yielded the Box-Behnken experiment variance table and the quadratic polynomial relating the response value to the independent variable, with the following results.

[0077] Table 4. Analysis of Variance of the Box-Behnken Trial

[0078]

[0079] Note: The difference was extremely significant (P < 0.01); the difference was significant (P < 0.05).

[0080] Obtain the quadratic polynomial relating the response value to the independent variable:

[0081] ;

[0082] In the formula: Y is the predicted response value, i.e., biomass, and the colony count is calculated as 10. 7 The inoculum is expressed as CFU / g; X1 is the inoculum amount (%), X2 is the culture temperature (°C), and X3 is the culture time (h).

[0083] As shown in the table above, the model R 2 =0.9147>0.9, indicating that the model fits well and the linear relationship between the independent variable and the response value reaches a significant level. The optimal fermentation parameters of Bacillus subtilis subsp.subtilis CEGF012 can be determined using this regression equation.

[0084] according to Figure 3 , Figure 4 and Figure 5 From the response surface and contour lines, we can see that:

[0085] When the culture temperature remains constant, the biomass of Bacillus subtilis subsp. subtilis CEGF012 will show a trend of first increasing and then decreasing as the inoculum size and time increase.

[0086] When the culture time remained constant, the biomass of Bacillus subtilis subsp. subtilis CEGF012 showed a trend of increasing and then decreasing with increasing temperature and inoculum size;

[0087] When the inoculum amount remained constant, the biomass of Bacillus subtilis subsp. subtilis CEGF012 showed a trend of first increasing and then decreasing with increasing temperature and time.

[0088] Setting the first-order partial derivatives of each independent variable (X1, X2, X3) to 0, and differentiating the fitted system of three linear equations, we obtain the model's predicted optimal points as follows: inoculum size 6.76%, culture time 28.66 h, and culture temperature 36.86 °C. Substituting these values ​​into the regression equation, we obtain the theoretical biomass Y = 728.859 × 10⁻⁶ for Bacillus subtilis subsp. subtilis CEGF012. 7 CFU / g.

[0089] Example 4 Model Validation

[0090] The fermentation was carried out under the optimal fermentation conditions (inoculum size 6.76%, culture time 28.66 h, culture temperature 36.86 °C) obtained by the predicted model in Example 3. After fermentation, the biomass of Bacillus subtilis subsp. subtilis CEGF012 was measured to verify the reliability of the model prediction. The results are shown in the table below:

[0091]

[0092] As shown in the table above, when cultured under the optimal fermentation conditions determined by the model, the biomass of Bacillus subtilis subsp. subtilis CEGF012 showed a 91.7% agreement with the model's predictions. This indicates that the model's predictions are accurate and reliable, and can be used to determine the appropriate inoculum size, culture temperature, and culture time based on a preset biomass, or to predict the biomass of Bacillus subtilis subsp. subtilis CEGF012 after culture based on a preset inoculum size, culture temperature, and culture time.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A strain of Bacillus subtilis subsp. subtilis CEGF012, characterized in that, Its accession number is CCTCC NO: M 20241702, and its accession date is July 31, 2024.

2. A microbial agent, characterized in that, Includes Bacillus subtilis subsp.subtilis CEGF012 as described in claim 1.

3. A method for culturing Bacillus subtilis subsp. subtilis CEGF012, characterized in that, Includes the step of inoculating the seed culture of Bacillus subtilis subsp. subtilis CEGF012 as described in claim 1 into a solid culture medium for cultivation; The nitrogen source in the solid culture medium includes plant-derived recalcitrant protein with a protein content of 15% to 30%, wherein the mass ratio of carbon source to nitrogen source is 50 to 83: 17 to 50.

4. The cultivation method as described in claim 3, characterized in that, The mass ratio of carbon source to nitrogen source in the solid culture medium is 50~75:25~50.

5. The cultivation method according to any one of claims 3 or 4, characterized in that, Bacillus subtilis subsp. subtilis CEGF012 seed culture was prepared according to the following method: The purified Bacillus subtilis subsp. subtilis CEGF012 was inoculated into LB liquid medium at a 2% inoculum and revived and propagated for 3 generations to obtain the Bacillus subtilis subsp. subtilis CEGF012 seed culture.

6. The cultivation method as described in claim 5, characterized in that, Based on the pre-defined biomass of Bacillus subtilis subsp. subtilis CEGF012, the inoculum size, culture temperature, and culture time were determined according to the following prediction model: ； In the formula: Y is the preset biomass, based on a colony count of 10-1. 7 CFU / g; X1 is the inoculum amount, expressed as a percentage; X2 is the culture temperature, in °C; X3 is the culture time, in h.

7. The cultivation method as described in claim 6, characterized in that, The culture conditions were as follows: the inoculum size of Bacillus subtilis subsp. subtilis CEGF012 seed culture was 4%~10%, the culture temperature was 35~40℃, and the culture time was 24~36h.

8. The cultivation method as described in claim 7, characterized in that, The culture conditions were as follows: the inoculum size of Bacillus subtilis subsp. subtilis CEGF012 seed culture was 6%~7%, the culture temperature was 36~37℃, and the culture time was 28~29h.

9. The cultivation method as described in claim 8, characterized in that, The mass ratio of carbon source to nitrogen source in the culture medium is 1:1.

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